Bottom Line:
On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain.At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization.Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures.

Affiliation: Department of Pharmacology, University of Vermont, Burlington, VT 05405 Department of Medicine, University of Vermont, Burlington, VT 05405, USA.

ABSTRACTThe expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress.

Figure 5: GFP-MLCK rings appear to be associated with discrete membrane structures at the base of the cell. (A) Stills taken of cells expressing GFP-MLCK (green) and RFP-farnesyl (red) for different times (in min) after hypotonic treatment. Different angled arrows mark different MLCK rings displayed in the following two panels. Scale bars are 15 μm. (B) Sequential stills of the ring marked by an arrow in the upper right corner of panel A from just before to 2.5 min after hypotonic treatment. The top row shows GFP-MLCK (green) and RFP-farnesyl (red). The middle row shows signal from MLCK, and the bottom row shows signal from farnesyl. The arrow indicates a time (1.2 min after hypotonic exposure) when both MLCK and some of the membrane marker appear as rings. Scale bars are 5 μm. (C) Sequential stills of the ring marked by an arrow in the upper left corner of panel A from 0.7 min to 3.5 min after hypotonic treatment. The top row shows GFP-MLCK (green) and RFP-farnesyl (red). The middle row shows MLCK signal, and the bottom row shows farnesyl signal. The arrow indicates a time (1.8 min) when the lumen of the MLCK ring is filled with membrane marker. Scale bars are 5 μm. (D) Stills from live imaging of cells transfected with GFP-MLCK (green) exposed to exogenous FM4-64X (red) for 2 min during hypotonic treatment and then washed out for the remaining recovery. An arrow marks an area where membrane dye has been internalized, such that GFP-MLCK signal colocalizes with an apparent FM4-64X ring. The adjacent panels depict magnified images of the MLCK signal for subsequent times. Over time, the MLCK ring appears to become a patch. The scale bar is 15 μm.

Mentions:
Given the possibility that MLCK localized to sites of membrane retrieval following swelling, we asked whether swelling-induced rings and patches at the cell base were associated with membrane structures and internalized membrane markers. We therefore performed live imaging of cells expressing GFP-MLCK and RFP-farnesyl, subjected them to hypotonic challenge, and monitored structures at the base as they formed and disappeared (Figure 5A). We found that MLCK rings could transiently be associated with farnesyl-labeled membrane, either colocalizing with farnesyl rings or encircling a lumen of membrane signal (Figure 5, B and C; Supplemental Figure S9). By contrast, detection of specific farnesyl signal in MLCK patches was not apparent, perhaps because of low signal (Supplemental Figure S10A).

Figure 5: GFP-MLCK rings appear to be associated with discrete membrane structures at the base of the cell. (A) Stills taken of cells expressing GFP-MLCK (green) and RFP-farnesyl (red) for different times (in min) after hypotonic treatment. Different angled arrows mark different MLCK rings displayed in the following two panels. Scale bars are 15 μm. (B) Sequential stills of the ring marked by an arrow in the upper right corner of panel A from just before to 2.5 min after hypotonic treatment. The top row shows GFP-MLCK (green) and RFP-farnesyl (red). The middle row shows signal from MLCK, and the bottom row shows signal from farnesyl. The arrow indicates a time (1.2 min after hypotonic exposure) when both MLCK and some of the membrane marker appear as rings. Scale bars are 5 μm. (C) Sequential stills of the ring marked by an arrow in the upper left corner of panel A from 0.7 min to 3.5 min after hypotonic treatment. The top row shows GFP-MLCK (green) and RFP-farnesyl (red). The middle row shows MLCK signal, and the bottom row shows farnesyl signal. The arrow indicates a time (1.8 min) when the lumen of the MLCK ring is filled with membrane marker. Scale bars are 5 μm. (D) Stills from live imaging of cells transfected with GFP-MLCK (green) exposed to exogenous FM4-64X (red) for 2 min during hypotonic treatment and then washed out for the remaining recovery. An arrow marks an area where membrane dye has been internalized, such that GFP-MLCK signal colocalizes with an apparent FM4-64X ring. The adjacent panels depict magnified images of the MLCK signal for subsequent times. Over time, the MLCK ring appears to become a patch. The scale bar is 15 μm.

Mentions:
Given the possibility that MLCK localized to sites of membrane retrieval following swelling, we asked whether swelling-induced rings and patches at the cell base were associated with membrane structures and internalized membrane markers. We therefore performed live imaging of cells expressing GFP-MLCK and RFP-farnesyl, subjected them to hypotonic challenge, and monitored structures at the base as they formed and disappeared (Figure 5A). We found that MLCK rings could transiently be associated with farnesyl-labeled membrane, either colocalizing with farnesyl rings or encircling a lumen of membrane signal (Figure 5, B and C; Supplemental Figure S9). By contrast, detection of specific farnesyl signal in MLCK patches was not apparent, perhaps because of low signal (Supplemental Figure S10A).

Bottom Line:
On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain.At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization.Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures.

Affiliation:
Department of Pharmacology, University of Vermont, Burlington, VT 05405 Department of Medicine, University of Vermont, Burlington, VT 05405, USA.

ABSTRACTThe expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress.